To open this file as a Word document, click here.

To open this file as a PDF, click here.

TUNEL Protocol using TACS TdT reagents

1         Fix 3.7% Formaldehyde in 1X PBS 30 min RT

• Add FRESH fix for 4 more hours
• 1X PBS wash 20 min
• 1X PBS wash 20 min

2         Infiltrate for OCT embedding

• 5% sucrose/1X PBS 30 min
• 5% sucrose/1X PBS 30 min
• 5% sucrose/1X PBS 30 min
• 30% sucrose/1X PBS 30 min or O/N 4°C
• 30% sucrose/1X PBS:OCT 1:1 4 hrs or O/N 4°C

3         Embed in OCT

• 100% OCT and freeze

4         Section

• 12-14 mm 2 PER SLIDE
• Dry on warmer (50°C) 30 min – 2 hrs
• Outline with PapPen

5         Permeablize tissue (Neuropore shelf of 4°C)

• Wash in 1X PBS in coppin jar
• Add 50 mL Neuropore reagent on each section in humidified chamber 75 min RT
• Wash 1X PBS 2 min
• Wash 1X PBS 2 min

LABEL MIX/1 SECTION 1mL TdT-dNTP           -20°C III 1mL Ion                        -20°C III 1mL TdT enzyme          -20°C II 50ml 1X label buffer      4°C

6         Label with TdT

• Incubate in 1X TdT Label Buffer 5 min
• Add 50 mL label mix 1 hr at 37°C (humidified)

7         Stop Reaction

• Incubate in copin jar full of 1X stop buffer 5 min
• Wash 2 min 1X PBS
• Wash 2 min 1X PBS

StrepAvidin-FITC mix 2 mL StrepA-F 400 mL 1X PBS 0.5 mL TO-PRO-3

8         StrepAvidin-(FITC) label (4°C fridge)

• 50mL StrepA-F on each section IN THE DARK 20 min
• Wash in copin jar full of 1X PBS, 0.1% Tween-20 2 min
• Wash again 2 m in